Language：English   Publishing type：Research paper (scientific journal)   Publisher：Scientific Reports  

We characterized the existence of O-β(1,4)-GlcNAc polymers (β1,4GNP) that were anchored on the O-linked glycosylation sites of shrimp thrombospondin (pmTSP-II). There were five putative β1,4GNP linkages on the epithelial growth factor-like domain of pmTSP-II. Antibody against O-β-GlcNAc (CTD110.6) was used to prove the existence of linear and complex β1,4GNP. The antibody well reacted with linear chito-triose, -tetraose and -pentaose conjugated with phosphatidylethanolamine lipid. The immunoreactivity could also be detected with a complex β1,4GNP within pmTSP-II (at MW > 250 kDa). Upon denaturing the protein with SDS-PAGE buffer, the size of pmTSP-II was shifted to be 250 kDa, approximately 2.5 folds larger than the deduced molecular mass of pmTSP-II (110 kDa), suggesting additional association of pmTSP-II apart from its known disulfide bridging. This was confirmed by chitinase digestion on pmTSP-II protein leading to the subsequent smaller protein bands at 110–170 kDa in time- and concentration-dependent manners. These bands well reacted with CTD110.6 antibody and disappeared after extensive chitinase hydrolysis. Together, we believe that β1,4GNP on pmTSP-II serve the function in an inter-chain association to provide structural architecture of egg extracellular matrix, a novel function of pmTSP-II in reproductive biology.

DOI： 10.1038/s41598-022-11873-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Glycoconjugate Journal  

Gangliosides are important components of the membrane and are involved in many biological activities. St8sia5 is an α2,8-sialyltransferase involved in ganglioside synthesis, and has three isoforms. In this study, we analyzed the features of three isoforms, St8sia5-S, -M, and -L that had not been analyzed, and found that only St8sia5-L was localized in the Golgi, while the majority of St8sia5-M and -S were localized in the ER. The localization of Golgi of St8sia5 depended on the stem region. In addition, the incorporation of exogenous GD3 was upregulated only in St8sia5-L expressing cells. Taken together, the localization of St8sia5 is important for the activity of the enzyme.

DOI： 10.1007/s10719-021-10034-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Scientific Reports  

Vertebrate CMP-sialic acid synthetase (CSS), which catalyzes the synthesis of CMP-sialic acid (CMP-Sia), consists of a 28 kDa-N-domain and a 20 kDa-C-domain. The N-domain is known to be a catalytic domain; however, the significance of the C-domain still remains unknown. To elucidate the function of the C-domain at the organism level, we screened the medaka TILLING library and obtained medaka with non-synonymous mutations (t911a), or single amino acid substitutions of CSS, L304Q, in the C-domain. Prominently, most L304Q medaka was lethal within 19 days post-fertilization (dpf). L304Q young fry displayed free Sia accumulation, and impairment of sialylation, up to 8 dpf. At 8 dpf, a marked abnormality in ventricular contraction and skeletal myogenesis was observed. To gain insight into the mechanism of L304Q-induced abnormalities, L304Q was biochemically characterized. Although bacterially expressed soluble L304Q and WT showed the similar Vmax/Km values, very few soluble L304Q was detected when expressed in CHO cells in sharp contrast to the WT. Additionally, the thermostability of various mutations of L304 greatly decreased, except for WT and L304I. These results suggest that L304 is important for the stability of CSS, and that an appropriate level of expression of soluble CSS is significant for animal survival.

DOI： 10.1038/s41598-021-01715-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Biological Chemistry  

Sialic acid (Sia)-binding immunoglobulin-like lectin 7 (Siglec-7) is an inhibitory receptor primarily expressed on natural killer (NK) cells and monocytes. Siglec-7 is known to negatively regulate the innate immune system through Sia binding to distinguish self and nonself; however, a counterreceptor bearing its natural ligand remains largely unclear. Here, we identified a counter-receptor of Siglec-7 using K562 hematopoietic carcinoma cells presenting cell surface ligands for Siglec-7. We affinity-purified the ligands using Fc-ligated recombinant Siglec-7 and diSia-dextran polymer, a strong inhibitor for Siglec-7. We then confirmed the counter-receptor for Siglec-7 as leukosialin (CD43) through mass spectrometry, immunoprecipitation, and proximity labeling. Additionally, we demonstrated that the cytotoxicity of NK cells toward K562 cells was suppressed by overexpression of leukosialin in a Siglec-7-dependent manner. Taken together, our data suggest that leukosialin on K562 is a counter-receptor for Siglec-7 on NK cells and that a cluster of the Sia-containing glycan epitope on leukosialin is key as trans-ligand for unmasking the cis-ligand.

DOI： 10.1016/j.jbc.2021.100477

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PLoS ONE  

ST8SIA2 is an important molecule regulating expression of the phenotype involved in schizophrenia. Lowered promoter activity of the ST8SIA2 gene is considered to be protective against schizophrenia by conferring tolerance to psychosocial stress. Here, we examined the promoter-type composition of anatomically modern humans (AMHs) and archaic humans (AHs; Neanderthals and Denisovans), and compared the promoter activity at the population level (population promoter activity; PPA) between them. In AMHs, the TCT-type, showing the second lowest promoter activity, was most prevalent in the ancestral population of non-Africans. However, the detection of only the CGT-type from AH samples and recombination tracts in AH sequences showed that the CGT- and TGT-types, exhibiting the two highest promoter activities, were common in AH populations. Furthermore, interspecies gene flow occurred into AMHs from AHs and into Denisovans from Neanderthals, influencing promoter-type compositions independently in both AMHs and AHs. The difference of promoter-type composition makes PPA unique in each population. East and Southeast Asian populations show the lowest PPA. This results from the selective increase of the CGC-type, showing the lowest promoter activity, in these populations. Every non-African population shows significantly lower PPA than African populations, resulting from the TCT-type having the highest prevalence in the ancestral population of non-Africans. In addition, PPA reduction is also found among subpopulations within Africa via a slight increase of the TCT-type. These findings indicate a trend toward lower PPA in the spread of AMHs, interpreted as a continuous adaptation to psychosocial stress arising in migration. This trend is considered as genetic tuning for the evolution of collective brains. The inferred promoter-type composition of AHs differed markedly from that of AMHs, resulting in higher PPA in AHs than in AMHs. This suggests that the trend toward lower PPA is a unique feature in AMH spread.

DOI： 10.1371/journal.pone.0259897

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioscience, Biotechnology and Biochemistry  

Sialic acids (Sias) are an outermost-situated sugar of glycoproteins and glycolipids to play important roles in various biological phenomena. They are often modified by additional substituents, such as O-acetyl group, to display more than 50 different structures in nature. Of those modified Sia, nothing is known about the occurrence and biological functions of sulfated Sias (SiaSs) in mammals. To elucidate the significance of sialic acid sulfation, we investigated various mammalian-cultured cell lines for the expression of SiaS using the specific antibody 3G9. First, SiaS is expressed in a cell line-dependent and a cell density-dependent manner. Second, in CHO cells, the expression of SiaS is reversibly induced by treatment with the antibiotic G418. Taken together, the expression of SiaS is changed by intrinsic and extrinsic factors in mammalian cells. This is the first demonstration of regulated expression of SiaS.

DOI： 10.1080/09168451.2020.1792763

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.14952/seikagaku.2020.920349

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Bacterial sialidases are widely used to remove sialic acid (Sia) residues from glycans. Most of them cleave the glycosides of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) under acidic pHs; however, currently available bacterial sialidases had no activity to the glycosides of deaminoneuraminic acid (Kdn). In this study, we found a novel sialidase from Sphingobacterium sp. strain HMA12 that could cleave any of the glycosides of Neu5Ac, Neu5Gc, and Kdn. It also had a broad linkage specificity, i.e., α2,3-, α2,6-, α2,8-, and α2,9-linkages, and the optimal pH at neutral ranges, pH 6.5–7.0. These properties are particularly important when sialidases are applied for in vivo digestion of the cell surface sialosides under physiological conditions. Interestingly, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), which is a transition state analog-based inhibitor, competitively inhibited the enzyme-catalyzed reaction for Kdn as well as for Neu5Ac, suggesting that the active site is common to the Neu5Ac and Kdn residues. Taken together, this sialidase is versatile and useful for the in vivo research on sialo-glycoconjugates.

DOI： 10.1016/j.bbrc.2019.12.079

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Neuroscience  

The extracellular glycan polysialic acid linked to neural cell adhesion molecule (PSA-NCAM) is principally expressed in the developing brain and the adult neurogenic regions. Although colocalization of PSA-NCAM with cholecystokinin (CCK) was found in the adult brain, the role of PSA-NCAM remains unclear. In this study, we aimed to elucidate the functional significance of PSA-NCAM in the CA1 region of the male mouse hippocampus. Combined fluorescence in situ hybridization and immunohistochemistry showed that few vesicular glutamate transporter 3-negative/CCK-positive (VGluT3 –/CCK +) cells were colocalized with PSA-NCAM, but most of the VGluT3 +/ CCK + cells were colocalized with PSA-NCAM. The somata of PSA-NCAM +/CCK + cells were highly innervated by serotonergic boutons than those of PSA-NCAM –/CCK + cells. The expression ratios of 5-HT3A receptors and p11, a serotonin receptor-interacting protein, were higher in PSA-NCAM +/CCK + cells than in PSA-NCAM –/CCK + cells. Pharmacological digestion of PSA-NCAM impaired the efficacy of antidepressant fluoxetine (FLX), a selective serotonin reuptake inhibitor, but not the efficacy of benzodiazepine anxiolytic diazepam. A Western blot showed that restraint stress decreased the expressions of p11 and mature brain-derived neurotrophic factor (BDNF), and FLX increased them. Interestingly, the FLX-induced elevation of expression of p11, but not mature BDNF, was impaired by the digestion of PSA-NCAM. Quantitative reverse transcription-polymerase chain reaction showed that restraint stress reduced the expression of polysialyltransferase ST8Sia IV and FLX elevated it. Collectively, PSA-NCAM colocalized with VGluT3 +/CCK + cells in the CA1 region of the hippocampus may play a unique role in the regulation of antidepressant efficacy via the serotonergic pathway.

DOI： 10.1523/JNEUROSCI.1779-19.2019

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

<p>Sialic acid (Sia) is known to present as a monosialyl residue at a non-reducing terminal end of glycan in nature. In some cases, Sias occur as polymerized sialyl structures, di/oligo/polysialic acids (diSia/oligoSia/polySias). Here, we focus on the structure, function and biological significance of diSia/oligoSia/polySias. Following the definition of di/oligo/polySia structures and establishment of chemical and immunochemical detection methods, we analyzed these structures and their function based on a set of working hypotheses. So far, the biological meanings of degree of polymerization (DP) were shown and they lead to the change the concept of polySia. In addition, this research is applicable in the study of diseases such as mental disorder and cancer. However, research on the biological significance of the diversity in di/oligo/polySia structures is still ongoing.</p>

DOI： 10.4052/tigg.1915.2sj

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

<p>Sialic acid (Sia) is known to present as a monosialyl residue at a non-reducing terminal end of glycan in nature. In some cases, Sias occur as polymerized sialyl structures, di/oligo/polysialic acids (diSia/oligoSia/polySias). Here, we focus on the structure, function and biological significance of diSia/oligoSia/polySias. Following the definition of di/oligo/polySia structures and establishment of chemical and immunochemical detection methods, we analyzed these structures and their function based on a set of working hypotheses. So far, the biological meanings of degree of polymerization (DP) were shown and they lead to the change the concept of polySia. In addition, this research is applicable in the study of diseases such as mental disorder and cancer. However, research on the biological significance of the diversity in di/oligo/polySia structures is still ongoing.</p>

DOI： 10.4052/tigg.1915.2SE

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Glycoscience: Basic Science to Applications: Insights from the Japan Consortium for Glycobiology and Glycotechnology (JCGG)  

DOI： 10.1007/978-981-13-5856-2_12

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.56.422

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DOI： 10.14952/SEIKAGAKU.2017.890634

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.55.22

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Glycobiology  

Polysialic acid (polySia) is a linear polymer of sialic acid that modifies neural cell adhesion molecule (NCAM) in the vertebrate brain. PolySia is a large and exclusive molecule that functions as a negative regulator of cell-cell interactions. Recently, we demonstrated that polySia can specifically bind fibroblast growth factor 2 (FGF2) and BDNF; however, the protective effects of polySia on the proteolytic cleavage of these proteins remain unknown, although heparin/heparan sulfate has been shown to impair the cleavage of FGF2 by trypsin. Here, we analyzed the protective effects of polySia on the proteolytic cleavage of FGF2 and proBDNF/BDNF. We found that polySia protected intact FGF2 from tryptic activity via the specific binding of extended polySia chains on NCAM to FGF2. Oligo/polySia also functioned to impair the processing of proBDNF by plasmin via binding of oligo/polySia chains on NCAM. In addition, the polySia structure synthesized by mutated polysialyltransferase, ST8SIA2/STX(SNP7), which was previously identified from a schizophrenia patient, was impaired for these functions compared with polySia produced by normal ST8SIA2. Taken together, these data suggest that the protective effects of polySia toward FGF2 and proBDNF may be involved in the regulation of the concentrations of these neurologically active molecules.

DOI： 10.1093/glycob/cwv049

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Neuroscience Research  

Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). Neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) is one of the major protein components of neuronal DRM. To determine the cellular function of NAP-22, interacting proteins were screened with an immunoprecipitation assay, and calcineurin (CaN) was detected. Further studies with NAP-22 prepared from DRM and CaN expressed in bacteria showed the binding of these proteins and a dose-dependent inhibitory effect of the NAP-22 fraction on the phosphatase activity of CaN. On the other hand, NAP-22 expressed in bacteria showed low binding to CaN and a weak inhibitory effect on phosphatase activity. To solve this discrepancy, identification of a nonprotein component that modulates CaN activity in the DRM-derived NAP-22 fraction was attempted. After lyophilization, a lipid fraction was extracted with chloroform/methanol. The lipid fraction showed an inhibitory effect on CaN without NAP-22, and further fractionation of the extract with thin-layer chromatography showed the presence of several lipid bands having an inhibitory effect on CaN. The mobility of these bands coincided with that of authentic ganglioside (GM1a, GD1a, GD1b, and GT1b), and authentic ganglioside showed an inhibitory effect on CaN. Treatment of lipid with endoglycoceramidase, which degrades ganglioside to glycochain and ceramide, caused a diminution of the inhibitory effect. These results show that DRM-derived NAP-22 binds several lipids, including ganglioside, and that ganglioside inhibits the phosphatase activity of CaN.

DOI： 10.1002/jnr.23599

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Biological Chemistry  

As acidic glycocalyx on primary mouse microglial cells and a mouse microglial cell line Ra2, expression of polysialic acid (polySia/PSA), a polymer of the sialic acid Neu5Ac (N-acetylneuraminic acid), was demonstrated. PolySia is known to modulate cell adhesion, migration, and localization of neurotrophins mainly on neural cells. PolySia on Ra2 cells disappeared very rapidly after an inflammatory stimulus. Results of knockdown and inhibitor studies indicated that rapid surface clearance of polySia was achieved by secretion of endogenous sialidase Neu1 as an exovesicular component. Neu1-mediated polySia turnover was accompanied by the release of brain-derived neurotrophic factor normally retained by polySia molecules. Introduction of a single oxygen atom change into polySia by exogenous feeding of the non-neural sialic acid Neu5Gc (N-glycolylneuraminic acid) caused resistance to Neu1-induced polySia turnover and also inhibited the associated release of brain-derived neurotrophic factor. These results indicate the importance of rapid turnover of the poly-Sia glycocalyx by exovesicular sialidases in neurotrophin regulation.

DOI： 10.1074/jbc.M115.638759

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biomolecules  

The 70-kDa heat shock protein (Hsp70), one of the major stress-inducible molecular chaperones, is localized not only in the cytosol, but also in extracellular milieu in mammals. Hsp70 interacts with various cell surface glycolipids including sulfatide (3'-sulfogalactosphingolipid). However, the molecular mechanism, as well as the biological relevance, underlying the glycolipid-Hsp70 interaction is unknown. Here we report that sulfatide promotes Hsp70 oligomerization through the N-terminal ATPase domain, which stabilizes the binding of Hsp70 to unfolded protein in vitro. We find that the Hsp70 oligomer has apparent molecular masses ranging from 440 kDa to greater than 669 kDa. The C-terminal peptide-binding domain is dispensable for the sulfatide-induced oligomer formation. The oligomer formation is impaired in the presence of ATP, while the Hsp70 oligomer, once formed, is unable to bind to ATP. These results suggest that sulfatide locks Hsp70 in a high-affinity state to unfolded proteins by clustering the peptide-binding domain and blocking the binding to ATP that induces the dissociation of Hsp70 from protein substrates.

DOI： 10.3390/biom5020958

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Biological Chemistry  

Mammalian sperm acquire fertility through a functional maturation process called capacitation, where sperm membrane molecules are drastically remodeled. In this study, we found that a wheat germ agglutinin (WGA)-reactive protein on lipid rafts, named WGA16, is removed from the sperm surface on capacitation. WGA16 is a prostate-derived seminal plasma protein that has never been reported and is deposited on the sperm surface in themalereproductive tract. BasedonproteinandcDNAsequences for purified WGA16, it is a homologue of human zymogen granule protein16(ZG16) belonging to the Jacalin-related lectin (JRL) family in crystal and primary structures. A glycan array shows that WGA16 binds heparin through a basic patch containing Lys-53/ Lys-73 residues but not the conventional lectin domain of the JRL family. WGA16 is glycosylated, contrary to other ZG16 members, and comparative mass spectrometry clearly shows its unique N-glycosylation profile among seminal plasma proteins. It has exposed GlcNAc and GalNAc residues without additional Gal residues. The GlcNAc/GalNAc residues can work as binding ligands foraspermsurface galactosyltransferase,whichactually galactosylatesWGA16in situ in the presence of UDP-Gal. Interestingly, surface removal of WGA16 is experimentally induced by either UDPGal or heparin. In the crystal structure, N-glycosylated sites and a potential heparin-binding site face opposite sides. This geography of two functional sites suggest that WGA16 is deposited on the sperm surface through interaction between its N-glycans and the surface galactosyltransferase, whereas its heparin-binding domain may be involvedinbindingto sulfatedglycosaminoglycansinthefemaletract, enabling removal ofWGA16from the sperm surface.

DOI： 10.1074/jbc.M114.635268

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DOI： 10.3109/10409238.2014.976606

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DOI： 10.1074/jbc.M113.496224

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DOI： 10.1093/glycob/cws132

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DOI： 10.3389/fncel.2013.00061

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DOI： 10.1093/glycob/cwr155

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1351/PAC-CON-11-12-10

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DOI： 10.1093/glycob/cwr081

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

Polysialic acid (polySia), particularly α2,8-linked polyNeu5Ac (DP=8∼400), is a unique polymer that spatio-temporally modifies neural cell adhesion molecule (NCAM), predominantly in embryonic brains. PolySia is considered to function as a negative regulator of adhesion between cells, and between cells and extracellular matrix due to its extremely polyanionic nature and steric hindrance. Through its anti-adhesive property and spatio-temporal expression, polySia is involved in cell migration, neural outgrowth, axonal guidance, synaptic plasticity, and the development of normal neural circuits and neurogenesis. Due to improvements of polySia-detection methods, it has been shown that polySia expression persists in the hippocampus and olfactory bulb of adults, where neurogenesis is ongoing; however, the functions of polySia, particularly in adult brains, remain incompletely resolved. Recently, we demonstrated that polySia functions not only as an anti-adhesive molecule, but also as a reservoir for specific bioactive molecules, such as neurotrophic factors, neurotransmitters, and growth factors, which regulate neural function. In addition, studies have revealed that the enzymatic activity of STX, which is involved in the synthesis of polySia, derived from schizophrenic patient is low and that the product of the enzyme, polySia on NCAM is impaired with respect to both quantity and quality. Notably, impaired polySia-NCAM cannot effectively retain bioactive molecules, including BDNF, dopamine, and FGF2. Taken together, the impairment of polySia might influence the retention function of polySia and lead to the development of psychiatric disorders, such as schizophrenia, which are reported to express either lower or elevated amounts of polySia compared with normal brains.

DOI： 10.4052/tigg.23.221

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DOI： 10.1074/jbc.M111.221143

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DOI： 10.1016/j.imlet.2010.06.007

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DOI： 10.1093/glycob/cwq030

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DOI： 10.11477/mf.2425100979

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DOI： 10.1016/S0076-6879(10)78010-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

Glycosaminoglycans (GAGs), large anionic glycopolymers, are the glycan portion of proteoglycans and are important components of the extracellular matrix. Recently we reported that polysialic acid, a polyanionic glycopolymer specific to the brain, binds neurotrophic factors to form a large complex. It is not clear whether GAGs also bind neurotrophic factors to form a large complex. In the present study, we demonstrate that a brain-derived neurotrophic factor (BDNF) dimer directly binds GAGs other than chondroitin and hyaluronic acid to form a large complex. Neurotrophin-3 showed similar GAG binding properties. Furthermore, BDNF, after forming a large complex with GAG, bound to the BDNF receptors tropomyosin-related kinase (Trk) B and p75 neurotrophin receptor (NTR). These findings suggest that GAGs function to produce a reservoir of BDNF and other neurotrophic factors, and may serve to regulate their local concentrations on the cell surface.

DOI： 10.1271/bbb.90637

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DOI： 10.1007/s10719-008-9161-5

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DOI： 10.1007/s10719-008-9184-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.s162_2

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DOI： 10.1093/glycob/cwn084

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DOI： 10.1016/j.biochi.2007.04.010

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DOI： 10.1093/glycob/cwm064

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DOI： 10.1016/j.bbrc.2007.04.197

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DOI： 10.1016/j.bbrc.2007.03.181

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DOI： 10.1016/j.bbrc.2007.01.139

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

Naturally occurring polysialic acids (polySias), a unique homopolymer of sialic acid (Sia) residues, have a large structural diversity, primarily arising from the diversities in Sia components as well as in the intersialyl linkages. The α2,8-linked polySia on the neural cell adhesion molecules (NCAM) is the most extensively studied and recognized as a regulator of cell-cell interactions during development and differentiation in vertebrate brain. However, little is known about the occurrence and functions of polySia other than the α2,8-linked polySia on NCAM. Recently, it has been shown that polySia occurs in nature more frequently than previously recognized, as analytical methods to detect polySia structures have been greatly improved. Using these methods, two different types of polySia, α2,8- and α2,9-linked polySia, have been discovered on the sea urchin sperm, although the α2,5<i>O_glycolyl</i>-linked polySia was already known in sea urchin egg. This is the first example of the co-localization of polySia with different intersialyl linkages in the same cell. The α2,9-linked polySia-containing glycoprotein of sea urchin sperm flagella was identified and named “flagellasialin”. The α2,9-linked polySia on flagellasialin is suggested as a regulator of intracellular Ca²⁺ and sperm motility. In addition, the α2,5<i>O_glycolyl</i>-linked polySia has recently been shown to increase intracellular pH and potentiate acrosome reaction of sea urchin sperm. These results indicate that these polySia structures in sea urchin gametes have new functions other than the negative regulator for the NCAM-mediated cell adhesion. In this review, we summarize the recent advances in structural and functional studies of polySia in sea urchin gametes.

DOI： 10.4052/tigg.19.85

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DOI： 10.1016/j.bbrc.2006.12.068

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CiNii Research

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DOI： 10.1093/jb/mvj200

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DOI： 10.1007/s10719-006-6734-z

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DOI： 10.1042/BJ20051459

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DOI： 10.1158/0008-5472.CAN-05-2615

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CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

CiNii Research

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CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

CiNii Research

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DOI： 10.1042/BJ20040299

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DOI： 10.1016/j.bmcl.2004.07.058

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CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

DOI： 10.4052/tigg.16.291

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CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

Sialic acids are commonly present as monosialyl residues at the non-reducing terminal end of glycoconjugates. In some cases, α2→8 linked di/oligosialic acid chains with DP 2 or 3 sialic acid residues are common structural units of gangliosides, and involved in various biological processes, such as cell adhesion, cell differentiation, signal transduction, and surface expression of stage specific antigen. In contrast, little attention has been paid to the occurrence and functions of such short sialyl di/oligomers on glycoproteins, while polysialic acid (DP≥8) in embryonic NCAM has been extensively studied as a regulator of cell adhesion in neurogenesis. As analytical methods to detect di/oligosialic acid structures have been improved, several glycoproteins containing di/oligo/polysialic acid chains have been identified. It is thus hypothesized that these di/oligosialic acid residues on glycoproteins may have similar important functions in common with those proposed for the gangliosides or may have new functions. Currently, several studies showing the importance of di/oligosialic acid-containing glycoproteins have emerged. In this review, recent advances in such studies of di/oligosialic acid residues on glycoproteins, including analytical methods, occurrence, biosynthetic pathways, and functions, are described.

DOI： 10.4052/tigg.16.331

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CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Biochemical Society  

Polysialoglycoprotein (PSGP) is a major cortical alveolus glycoprotein of rainbow trout eggs that is characterized by the attachment of a polysialic acid structure to its <i>O</i>-glycan chains. It has been demonstrated that the polysialic acid structure is synthesized by at least three sialyltransferases mostly localized in cortical alveoli. Here we have cloned a cDNA encoding the sialyltransferase, designated rtST6GaINAc, responsible for the transfer of the first sialic acid residue onto the <i>O</i>-glycan chain of PSGP. This enzyme belongs to the vertebrate ST6GaINAc II family, and is strongly expressed in ovaries. Of those <i>O</i>-glycoproteins tested as substrates, asialo-PSGP is the best substrate. These results indicate that rtST6GalNAc is the enzyme responsible for the sialylation of PSGP during oogenesis. Furthermore, the rtST6GalNAc mRNA is expressed throughout oogenesis, is down-regulated at the late yolk vesicle stage (May), and then up-regulated during vitellogenesis (until August). This developmental profile is highly similar to that of STL2, a cortical alveolus lectin, while it is quite different from that of PSGP, which is extensively expressed at the yolk vesicle stage and down-regulated at later stages. Thus, not all cortical alveolus components are transcribed concomitantly. This is the first description of a developmental change in the transcription of a glycosyltransferase during oogenesis.

DOI： 10.1093/jb/mvh106

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CiNii Research

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DOI： 10.1016/j.bmc.2004.02.009

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CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)  

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

CiNii Research

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DOI： 10.1074/jbc.M206046200

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：生化学工業株式会社  

DOI： 10.32285/glycoforum.06a4j

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SEIKAGAKU CORPORATION  

DOI： 10.32285/glycoforum.06a4

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Journal of Biological Chemistry  

Integrin α5β1, a major fibronectin receptor, functions in a wide variety of biological phenomena. We have found that α2-8-linked oligosialic acids with 5 ≤ degree of polymerization (DP) ≤ 7 occur on integrin α5 subunit of the human melanoma cell line G361. The integrin α5 subunit immunoprecipitated with anti-integrin α5 antibody reacted with the monoclonal antibody 12E3, which recognizes oligo/polysialic acid with DP ≥ 5 but not with the polyclonal antibody H.46 recognizing oligo/polysialic acid with DP ≥ 8. The occurrence of oligosialic acids was further demonstrated by fluorometric C7/C9 analysis on the immunopurified integrin α5 subunit. Oligosialic acids were also found in the α5 subunit of several other human cells such as foreskin fibroblast and chronic erythroleukemia K562 cells. These results suggest the ubiquitous modification with unique oligosialic acids occurs on the α 5 subunit of integrin α5β1. The adhesion of human melanoma G361 cells to fibronectin was mainly mediated by integrin α5β1. Treatment of cells with sialidase from Arthrobacter ureafaciens cleaving α2-3-, α2-6-, and α2-8-linked sialic acids inhibited adhesion to fibronectin. On the other hand, N-acetylneuraminidase II, which cleaves α2-3 and α2-6 but not α2-8 linkages, showed no inhibitory activity. After the loss of oligosialic acids, integrin α5β1 failed to bind to fibronectin-conjugated Sepharose, indicating that the oligosialic acid on the α5 subunit of integrin α5β 1 plays important roles in cell adhesion to fibronectin.

DOI： 10.1074/jbc.M011100200

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M104148200

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

Sialic acids, mainly <i>N</i>-acetylneuraminic acid, have been found during the development of a few insect species. In <i>Drosophila melanogaster</i> and the cicada <i>Philaenus spumarius</i> we detected polysialic acids, using lectins, antibodies, and various methods for the isolation and analysis of the monosaccharide subunits, in areas of neuronal development and in Malpighian tubules, respectively. However, sialic acids were not found in the adult animals of these species. Although the expression of these acidic sugars has unequivocally been demonstrated <i>in vivo</i>, their occurrence in insect cell cultures was, in most cases, not clearly demonstrated. The biosynthesis of sialic acids in cell cultures would be of great biotechnological significance, since the production of recombinant glycoproteins with mammalian type, complex, sialylated <i>N</i>-glycans using the baculovirus expression system would be of great benefit. From the studies dedicated to this problem it appears that the engineering of such glycans may only be possible by the expression of exogenous genes in insect cells. Several genes encoding sialic acid-metabolizing enzymes have therefore been transfected. The work on insect cells shows that sialic acids are not only restricted to the Deuterostomia branch of the animal kingdom but also occur in some Protostomia species, as in insects. This throws new light on the evolution of this acidic sugar.

DOI： 10.4052/tigg.13.507

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1023/A:1026589223811

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Journal of Biological Chemistry  

The pre-existence of α2→8-linked disialic acid (di-Sia) and oligosialic acid (oligo-Sia) structures with up to 7 Sia residues was shown to occur on a large number of brain glycoproteins, including neural cell adhesion molecules (N-CAMs), by two highly sensitive chemical methods (Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1998) Anal. Biochem. 261, 191- 197; Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1999) Anal Biochem. 266, 102-109). This unexpected finding was also confirmed using a newly developed antibody prepared using a copolymer of α2→8-linked N- acetylneuraminyl p-vinylbenzylamide and acrylamide as an immunogen and known antibodies whose immunospecificities were determined to be di- and oligo-Sia residues with defined chain lengths. The major significance of the new finding that di- and oligo-Sia chains exist on a large number of brain glycoproteins is 2-fold. First, it reveals a surprising diversity in the number and M(r) of proteins distinct from N-CAM that are covalently modified by these short sialyl glycotopes. Second, it suggests that synthesis of di- and/or oligo-Sia units may be catalyzed by α2→8-sialyltransferase(s) that are distinct from the known polysialyltransferases, STX and PST, which are partially responsible for polysialylation of N-CAM.

DOI： 10.1074/jbc.275.20.15422

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1017/s0967199400130321

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

Sialic acids are acidic, 9-carbon carboxylated monosaccharides, typically expressed on the outermost end of the sugar chains of membrane and secreted glycoconjugates. This particular location provides it accessibility reflected in its regulation of a multitude of cellular and molecular interactions. Amongst the diverse structural variations that occur, the commonest is <i>O</i>-acetylation at C-_{4, 7, 8} and₉ carbon positions. This diversity in <i>O</i>-acetylated sialic acid (<i>O</i>-AcSA) derivatives is attributed to their linkage to the underlying sugar chain, the carbon position that gets <i>O</i>-acetylated and nature of the glycoconjugates to which they are attached. Steady refinement of analytical techniques has allowed for identification of <i>O</i>-AcSA derivatives in several diseases leading to a shift in focus towards understanding the biological importance of this modification. The present review deals primarily with the recently evolved information regarding detection of altered <i>O</i>-acetylation in various pathophysiological conditions and their biological relevance. Particular attention is focused on two diverse diseases, namely, Acute Lymphoblastic Leukemia (ALL) and Visceral Leishmaniasis (VL) in which 9-<i>O</i> acetylated sialoglycoconjugates (9-<i>O</i>AcSGs) have proved to be effective biomarkers for monitoring their clinical status.

DOI： 10.4052/tigg.12.17

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

α2→8-Linked di- and oligosialic acid (diSia and oligoSia) chains with DP 2, 3 Sia residues are known to be common structural units of gangliosides, and to be involved in various biological processes, such as cell adhesion, cell differentiation, signal transduction, and surface expression of stage specific antigen. In contrast, little attention has been paid to the occurrence and functions of such short sialyl oligomers on glycoproteins. However, it has recently been shown that glycoproteins containing di- and oligoSia groups occur in nature more frequently than was ever recognized, as analytical methods to detect di- and oligoSia structures have improved. It is thus hypothesized that these di- and oligoSia moieties on glycoproteins may have similar important functions in common with those proposed for the gangliosides. In this review, we describe the recent advances in the study of di- and oligoSia residues on glycoproteins, including analytical methods, occurrence, functions, and biosynthetic pathways.

DOI： 10.4052/tigg.11.371

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CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.274.30.21369

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DOI： 10.1006/bbrc.1999.0686

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DOI： 10.1046/j.1432-1327.1999.00203.x

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DOI： 10.1006/abio.1998.2921

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Analytical Biochemistry  

A highly sensitive chemical method for detecting α2→8-linked oligo/polysialic acid (oligo/polySia) chains was developed, including (i) periodate oxidation, reduction with sodium borohydride, and subsequent acid hydrolysis, giving rise to C7 analogues and intact C9 compounds from nonreducing terminal and internal sialic acid residues, respectively; (ii) fluorescent labeling of these C7 and C9 compounds with 1,2-diamino-4,5- methylenedioxybenzene (DMB); and (iii) quantitation of these DMB derivatives on fluorometric high-performance liquid chromatography. As little as 1 ng of internal sialic acid residues of oligo/polySia chains, the existence of which indicates the presence of oligo/polySia structure, was detectable by this method. This fluorometric C7/C9 analysis was successfully applied to glycoproteins blotted onto a slit of polyvinylidene fluoride membranes and suggested the presence of some novel oligoSia-containing glycoproteins in pig embryonic brains.

DOI： 10.1006/abio.1998.2718

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DOI： 10.1074/jbc.273.5.2575

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Glycobiology  

The Pronase digestion of a 54K glycoprotein present in ovarian fluid of rainbow trout yielded a major glycopeptide. Carbohydrate compositional analysis revealed that this glycopeptide was likely to possess a single large N-glycan chain having low molecular weight oligomers of N-acetylneuraminic acid (oligoNeu5Ac). Structural studies of this glycopeptide revealed novel α2 → 8-linked disialylated poly-N-acetyllactosamine chains with Le(X) and I antigenic determinants on the N-linked tetraantennary core glycan. In our recent studies (Kitazume, S., Kitajima, K., Inoue, S., Inoue, Y. and Troy, F.A. (1994) J. Biol. Chem. 269, 10330-10340) we presented evidence that synthesis of α2 → 8-linked polysialic acid (polySia) chains is a two-step process in which chain initiation is catalyzed by an α2 → 8-sialyltransferase (α2 → 8-ST; initiase) that catalyzes synthesis of the first Sia α2 → 8-linkage, forming the disialic acid (diSia) unit, Siaα2 → 8-Siaα2 → 6-Gal-.Chain polymerization is then postulated to be catalyzed by a second enzyme, an α2 → 8-polyST ('polymerase') that converts the diSia units to polySia chains. The present structural studies leading to the discovery of α2 → 8-linked disialylated units that terminate poly-N-acetyllactosamine chains in an N-linked glycoprotein is further evidence in support of our hypothesis that more than one sialyltransferase activity is required for polySia chain synthesis and polymerization.

DOI： 10.1093/glycob/7.2.195

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DOI： 10.1016/j.jbc.2024.105712

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DOI： 10.7554/eLife.92342.

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DOI： 10.1016/j.bbrc.2023.01.010

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DOI： 10.3390/biom13010146

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A transition of sialic acid (Sia) species on GM3 ganglioside from N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc) takes place in mouse C2C12 myoblast cells during their differentiation into myotube cells. However, the meaning of this Sia transition remains unclear. This study thus aims to gain a functional insight into this phenomenon. The following lines of evidence show that the increased de novo synthesis of Neu5Gc residues in differentiating myoblast cells promotes adhesiveness of the cells, which is beneficial for promotion of differentiation. First, the Sia transition occurred even in the C2C12 cells cultured in serum-free medium, indicating that it happens through de novo synthesis of Neu5Gc. Second, GM3(Neu5Gc) was localized in myoblast cells, but not in myotube cells, and related to expression of the CMP-Neu5Ac hydroxylase (CMAH) gene. Notably, expression of CMAH precedes myotube formation not only in differentiating C2C12 cells, but also in mouse developing embryos. Since the myoblast cells were attached on the dish surface more strongly than the myotube cells, expression of GM3(Neu5Gc) may be related to the surface attachment of the myoblast cells. Third, exogenous Neu5Gc, but not Neu5Ac, promoted differentiation of C2C12 cells, thus increasing the number of cells committed to fuse with each other. Fourth, the CMAH-transfected C2C12 cells were attached on the gelatin-coated surface much more rapidly than the mock-cells, suggesting that the expression of CMAH promotes cell adhesiveness through the expression of Neu5Gc.

DOI： 10.1007/s10719-022-10049-9

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Sialylation, the final stage of post-translational modification of proteins, is achieved in the Golgi apparatus and is related to the malignant phenotype of cancer. Disialylation of ganglioside (GD3) by St8sia1 and polysialylation by St8sia2 and 4 have been shown to be related to malignant phenotypes; however, di/oligosialylation by St8sia6 is still unknown. In this study, we analyzed the malignant phenotype of St8sia6 and found that upregulation of St8sia6 in melanoma B16 cells increased anchorage-independent cell growth, which was not due to sialic acid cleavage by a sialidase. Moreover, unlike other sialyltransferases, St8sia6 localized to the endoplasmic reticulum (ER). We found that the localization to the Golgi apparatus could be regulated by swapping experiments using St8sia2; however, the malignant phenotype did not change. These data demonstrate that the enhancement of anchorage-independent cell growth by St8sia6 is not due to its localization of ER, but is due to the expression of the protein itself.

DOI： 10.1016/j.bbrc.2022.03.146

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In cancer cells, cell-surface sialylation is altered, including a change in oligo/polysialic acid (oligo/polySia) structures. Since they are unique and rarely expressed in normal cells, oligo/polySia structures may serve as promising novel biomarkers and targets for therapies. For the diagnosis and treatment of the disease, a precise understanding of the oligo/polySia structures in cancer cells is necessary. In this study, flow cytometric analysis and gene expression datasets were obtained from sixteen different cancer cell lines. These datasets demonstrated the ability to predict glycan structures and their sialylation status. Our results also revealed that sialylation patterns are unique to each cancer cell line. Thus, we can suggest promising combinations of antibody and cancer cell for glycan prediction. However, the precise prediction of minor glycans need to be further explored.

DOI： 10.3390/ijms23105569

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Schizophrenia is a serious psychiatric disorder that affects the social life of patients. Psychiatric disorders are caused by a complex combination of genetic (G) and environmental (E) factors. Polysialylation represents a unique posttranslational modification of a protein, and such changes in neural cell adhesion molecules (NCAMs) have been reported in postmortem brains from patients with psychiatric disorders. To understand the G × E effect on polysialylated NCAM expression, in this study, we performed precise measurements of polySia and NCAM using a disrupted-in-schizophrenia 1 (DISC1)-mutant mouse (G), a mouse model of schizophrenia, under acute stress conditions (E). This is the first study to reveal a lower number and smaller length of polySia in the suprachiasmatic nucleus of DISC1 mutants relative to those in wild-type (WT) mice. In addition, an analysis of polySia and NCAM responses to acute stress in five brain regions (olfactory bulb, prefrontal cortex, suprachiasmatic nucleus, amygdala, and hippocampus) revealed that the pattern of changes in these responses in WT mice and DISC1 mutants differed by region. These differences could indicate the vulnerability of DISC1 mutants to stress.

DOI： 10.3390/ijms23095207

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Polysialic acid (polySia, PSA) is a unique constituent of the glycocalyx on the surface of bacterial and vertebrate cells. In vertebrates, its biosynthesis is highly regulated, not only in quantity and quality, but also in time and location, which allows polySia to be involved in various important biological phenomena. Therefore, impairments in the expression and structure of polySia sometimes relate to diseases, such as schizophrenia, bipolar disorder, and cancer. Some bacteria express polySia as a tool for protecting themselves from the host immune system during invasion. PolySia is proven to be a biosafe material; polySia, as well as polySia-recognizing molecules, are key therapeutic agents. This review first comprehensive outlines the occurrence, features, biosynthesis, and functions of polySia and subsequently focuses on the related diseases.

DOI： 10.1016/j.mam.2020.100892

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Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat-induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate-Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.

DOI： 10.1172/JCI137681

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Surface expression of the common vertebrate sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia N-glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable Haemophilus influenzae (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like Cmah null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens.IMPORTANCE All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.

DOI： 10.1128/mBio.03226-20

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Siglecs are sialic acid (Sia)-binding immunoglobulin-like lectins; the majority of Siglecs functions as transmembrane receptors on the immune cells via Sia residues. Recently, a new Sia binding site in Siglec-7, termed site 2, where arginine (R) 67 was critical, was identified by computational modeling and biochemical analyses, relative to the primary Sia binding site, termed site 1, containing critical R124. Here, the presence of a new essential R94 residue, which is completely conserved among all identified Siglecs, was demonstrated. A mutation of R94 residue in Siglec-7 led to the disappearance of the Sia binding property, similar to a site 1 mutation (R124A). R94 is close to R67 in site 2, and site 2 mutations at either of them abolished the ligand-binding properties to both gangliosides and glycoproteins. These data suggest that, in addition to site 1, the conserved R residue among Siglecs in site 2 is another functional site.

DOI： 10.1016/j.bbrc.2020.10.023

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Polysialic acid (polySia/PSA) is a linear homopolymer of sialic acid (Sia) that primarily modifies the neural cell adhesion molecule (NCAM) in mammalian brains. PolySia-NCAM not only displays an anti-adhesive function due to the hydration effect, but also possesses a molecule-retaining function via a direct binding to neurologically active molecules. The quality and quantity of polySia determine the function of polySia-NCAM and are considered to be profoundly related to the maintenance of normal brain functions. In this study, to compare the structures of polySia-NCAM in brains of five different vertebrates (mammals, birds, reptiles, amphibians, and fish), we adopted newly developed combinational methods for the analyses. The results revealed that the structural features of polySia considerably varied among different species. Interestingly, mice, as a mammal, possess eminently distinct types of polySia, in both quality and quantity, compared with those possessed by other animals. Thus, the mouse polySia is of larger quantities, of longer and more diverse chain lengths, and of a larger molecular size with higher negative charge, compared with polySia of other species. These properties might enable more advanced brain function. Additionally, it is suggested that the polySia/Sia ratio, which likely reflects the complexity of brain function, can be used as a new promising index to evaluate the intelligence of different vertebrate brains.

DOI： 10.3390/ijms21228593

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Polysialic acid (polySia/PSA) is an anionic glycan polymer of sialic acid, and it mostly modifies the neural cell adhesion molecule (NCAM) in mammalian brains. Quality and quantity of the polySia of the polySia-NCAM is spatio-temporally regulated in normal brain development and functions, and their impairments are reported to be related to diseases, such as psychiatric disorders and cancers. Therefore, precise understanding of the state of polySia-NCAM structure would lead to the diagnosis of diseases for which their suitable evaluation methods are necessary. In this study, to develop these evaluation methods, structures of polySia-NCAM from mouse brains at six different developmental stages were analyzed by several conventional and newly developed methods. Integrated results of these experiments clearly demonstrated the existence of different types of polySia-NCAMs in developing brains. In addition, combinational analyses were shown to be useful for precise understanding of the quantity and quality of polySia, which can provide criteria for the diagnosis of diseases.

DOI： 10.3390/ijms21165892

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Siglec-7 is a human CD33-like siglec, and is localised predominantly on human natural killer (NK) cells and monocytes. Siglec-7 is considered to function as an immunoreceptor in a sialic acid-dependent manner. However, the underlying mechanisms linking sialic acid-binding and function remain unknown. Here, to gain new insights into the ligand-binding properties of Siglec-7, we carried out in silico analysis and site-directed mutagenesis, and found a new sialic acid-binding region (site 2 containing R67) in addition to the well-known primary ligand-binding region (site 1 containing R124). This was supported by equilibrium dialysis, STD-NMR experiments, and inhibition analysis of GD3-binding toward Siglec-7 using synthetic sialoglycoconjugates and a comprehensive set of ganglioside-based glycoconjugates. Our results suggest that the two ligand-binding sites are potentially controlled by each other due to the flexible conformation of the C-C' loop of Siglec-7.

DOI： 10.1038/s41598-020-64887-4

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ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), which is essential for brain development. Although previous studies reported that St8sia2-deficient mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variable phenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A genomic polymorphism and gene expression analysis in the flanking region revealed reduced expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on that of the 129 strain. This observation, along with the finding that administration of an IGF1R agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importance of gene expression level in the flanking regions of a targeted null allele having an effect on phenotype.

DOI： 10.1038/s41598-019-50006-5

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DOI： 10.1038/s41398-019-0529-z

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DOI： 10.1038/s41598-019-46240-6

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DOI： 10.1002/mrd.23122

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DOI： 10.1016/bs.accb.2018.09.003

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DOI： 10.1007/978-981-13-5856-2_2

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DOI： 10.1002/asia.201801269

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The emergence of zebrafish Danio rerio as a versatile model organism provides the unique opportunity to monitor the functions of glycosylation throughout vertebrate embryogenesis, providing insights into human diseases caused by glycosylation defects. Using a combination of chemical modifications, enzymatic digestion and mass spectrometry analyses, we establish here the precise glycomic profiles of eight individual zebrafish organs and demonstrate that the protein glycosylation and glycosphingolipid expression patterns exhibits exquisite specificity. Concomitant expression screening of a wide array of enzymes involved in the synthesis and transfer of sialic acids shows that the presence of organ-specific sialylation motifs correlates with the localized activity of the corresponding glycan biosynthesis pathways. These findings provide a basis for the rational design of zebrafish lines expressing desired glycosylation profiles.

DOI： 10.1038/s41467-018-06950-3

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DOI： 10.1007/s10719-018-9832-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

A number of loci are associated with highly heritable schizophrenia and the prevalence of this mental illness has had considerable negative fitness effects on human populations. Here we focused on one particular schizophrenia-associated gene that encodes a sialyl-transferase (ST8SIA2) and is expressed preferentially in the brain with the level being largely determined by three SNPs in the promoter region. It is suggested that the expression level of the ST8SIA2 gene is a genetic determinant of schizophrenia risk, and we found that a geographically differentiated non-risk SNP type (CGC-type) has significantly reduced promoter activity. A newly developed method for detecting ongoing positive selection was applied to the ST8SIA2 genomic region with the identification of an unambiguous sweep signal in a rather restricted region of 18 kb length surrounding the promoter. We also found that while the CGC-type emerged in anatomically modern humans in Africa over 100 thousand years ago, it has increased its frequency in Asia only during the past 20-30 thousand years. These findings support that the positive selection is driven by psychosocial stress due to changing social environments since around the last glacial maximum, and raise a possibility that schizophrenia extensively emerged during the Upper Paleolithic and Neolithic era.

DOI： 10.1371/journal.pone.0200278

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Polysialic acid (polySia) is mainly found as a modification of neural cell adhesion molecule (NCAM) in whole embryonic brains, as well as restricted areas of adult vertebrate brains, including the hippocampus. PolySia shows not only repulsive effects on NCAM-involved cell-cell interactions due to its bulky and hydrated properties, but also attractive effects on the interaction with neurologically active molecules, which exerts a reservoir function. Two different polysialyltransferases, ST8SIA2 and ST8SIA4, are involved in the synthesis of polySia chains; however, to date, the differences of the properties between polySia chains synthesized by these two enzymes remain unknown. In this study, to clarify this point, we first prepared polySia-NCAMs from HEK293 cells stably expressing ST8SIA4 and ST8SIA2, or ST8SIA2 (SNP-7), a mutant ST8SIA2 derived from a schizophrenia patient. The conventional sensitive chemical and immunological characterizations showed that the quantity and quality (structural features) of polySia are not so much different between ST8SIA4-and ST8SIA2-synthesized ones, apart from those of ST8SIA2 (SNP-7). Then, we assessed the homophilic and heterophilic interactions mediated by polySia-NCAM by adopting a surface plasmon resonance measurement as an in vitro analytical method. Our novel findings are as follows: (i) the ST8SIA2-and ST8SIA4-synthesized polySia-NCAMs exhibited different attractive and repulsive effects than each other; (ii) both polySia-and oligoSia-NCAMs synthesized by ST8SIA2 were able to bind polySia-NCAMs; (iii) the polySia-NCAM synthesized by a ST8SIA2 (SNP-7) showed markedly altered attractive and repulsive properties. Collectively, polySia-NCAM is suggested to simultaneously possess both attractive and repulsive properties that are highly regulated by the two polysialyltransferases.

DOI： 10.1093/glycob/cwx057

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

A new sialic acid (Sia)-containing glycopolymer-a fluorescent probe with high-density disialic acid (diSia) on the surface of polysaccharide dextran (diSia-Dex)-was synthesized as a key molecule to regulate the Sia recognition lectins, Siglecs, that are involved in the immune system. According to our original methods, diSia was synthesized by alpha-selective sialylation, and a dextran template possessing terminal acetylenes and amino groups was prepared. A diSia and a fluorescent molecule were subsequently introduced to surface-modified dextran by Huisgen reaction and amidation, respectively. The modulatory activity of Siglec7 was evaluated by using synthetic probes. DiSia-Dex showed high binding avidity toward Siglec7, with a KD value of 5.87 x 10(-10) M, and a high inhibitory activity for the interaction between Siglec7 and a ligand (GD3), with a IC50 value of 1.0 nM. Notably, diSia-Dex was able to release Siglec7 from the pre-existing Siglec7-GD3 complex, possibly due to its unique properties of a slow dissociation rate and a high association rate. Together, these data show that diSia-Dex can be widely applicable as a modulator of Siglec7 functions.

DOI： 10.1002/cbic.201600694

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A 250-kDa protein was isolated from fluid in the middle spermatic duct (MSD) of the blue crab (Portunus pelagicus). N-terminal and partial amino acid sequences revealed that this MSD-specific protein is highly similar to the plasma-enriched protein Alpha-2 macroglobulin (alpha 2M). The P. pelagicus ortholog (Pp alpha 2M) is a large glycoprotein possessing mannose and N-acetylglucosamine residues. Pp alpha 2M mRNA was detected in the spermatic duct, androgenic gland, and hematopoietic tissue, whereas the protein was primarily observed in the apical cytoplasm of MSD epithelium and in the matrix of the acrosome of MSD sperm; distally within spermatic duct, Pp alpha 2M was lost from the sperm membrane but remained in the sperm acrosome. These results suggest that Pp alpha 2M is expressed and glycosylated in the epithelium of spermatic ducts, secreted into MSD fluid, taken up by sperm in the MSD, and removed from the surface of sperm during its transit towards the female spermatheca. Given that Pp alpha 2M also exhibits protease inhibitor activity, we hypothesize that acrosome localized Pp alpha 2M may suppress premature acrosome reaction during post-testicular sperm maturation in this crab.

DOI： 10.1002/mrd.22817

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The neural cell adhesion molecule (NCAM) is modified by polysialic acid (polySia or PSA) in embryonic brains. In adult brains, polySia modification of NCAM is only observed in restricted areas where neural plasticity, remodeling of neural connections, or neural generation is ongoing although the amount of NCAM remains unchanged. Impairments of the polySia-expression and several single nucleotide polymorphisms (SNPs) of the polysialyltransferase (polyST) ST8SIA2 gene are reported to be associated with schizophrenia and bipolar disorder. Chlorpromazine (CPZ) is well-known as an agent for treating schizophrenia, and our hypothesis is that CPZ may affect the polySia expression or the gene expression of polySTs or NCAM. To test this hypothesis, we analyzed the effects of CPZ on the expression of polySia-NCAM on human neuroblastoma cell line, IMR-32 cells, by immunochemical and chemical methods. Interestingly, the cell surface expression of polySia, especially those with lower chain lengths, was significantly increased on the CPZ-treated cells, while mRNAs for polySTs and NCAM, and the amounts of total polySia-NCAM remained unchanged. The addition of brefeldin A, an inhibitor of endocytosis, suppressed the CPZ-induced cell surface polySia expression. In addition, polySia-NCAM was also observed in the vesicle compartment inside the cell. All these data suggest that the level of cell surface expression of polySia in IMR-32 is highly regulated and that CPZ changes the rate of the recycling of polySia-NCAM, leading to the up-regulation of polySia-NCAM on the cell surface. We also analyzed the effect of CPZ on polySia-expression in various brain regions in adult mice and found that CPZ only influenced the total amounts of polySia-NCAM in prefrontal cortex. These results suggest a brain-region-specific effect of CPZ on the expression of total polySia in mouse brain. Collectively, anti-schizophrenia agent CPZ consistently up-regulates the expression polySia at both cellular and animal levels.

DOI： 10.3390/ijms18061123

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Gangliosides (sialic acid-containing glycosphingolipids) help regulate many important biological processes, including cell proliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The structural diversity of gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecular species of a given ganglioside are suggested to have distinct biological properties and regulate specific and distinct biological events. Elucidation of the function of each molecular species is important and will provide new insights into ganglioside biology. Gangliosides are also suggested to be involved in skeletal muscle differentiation; however, the differential roles of ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of gangliosides (particularly GM3) during differentiation of mouse C2C12 myoblast cells and key roles played by distinct GM3 molecular species at each step of the process.

DOI： 10.1074/jbc.M116.771253

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

The occurrence and biological importance of sialic acid (Sia) and its metabolic enzymes in insects have been studied using Drosophila melanogaster. The most prominent feature of D. melanogaster CMP-Sia synthetase (DmCSS) is its Golgi-localization, contrasted with nuclear localization of vertebrate CSSs. However, it remains unclear if the Golgi-localization is common to other insect CSSs and why it happens. To answer these questions, Aedes aegypti (mosquito) CSS (AaCSS) and Tribolium castaneum (beetle) CSS (TcCSS) were cloned and characterized for their activity and subcellular localization. Our new findings show: (1) AaCSS and TcCSS share a common overall structure with DmCSS in terms of evolutionarily conserved motifs and the absence of the C-terminal domain typical to vertebrate CSSs; (2) when expressed in mammalian and insect cells, AaCSS and TcCSS showed in vivo and in vitro CSS activities, similar to DmCSS. In contrast, when expressed in bacteria, they lacked CSS activity because the N-terminal hydrophobic region appeared to induce protein aggregation; (3) when expressed in Drosophila S2 cells, AaCSS and TcCSS were predominantly localized in the ER, but not in the Golgi. Surprisingly, DmCSS was mainly secreted into the culture medium, although partially detected in Golgi. Consistent with these results, the N-terminal hydrophobic regions of AaCSS and TcCSS functioned as a signal peptide to render them soluble in the ER, while the N-terminus of DmCSS functioned as a membrane-spanning region of type II transmembrane proteins whose cytosolic KLK sequence functioned as an ER export signal. Accordingly, the differential subcellular localization of insect CSSs are distinctively more diverse than previously recognized.

DOI： 10.1093/glycob/cww128

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

An allene monomer containing an alpha(2,8) disialic acid was polymerized by a pi-allyl nickel complex with an azido group to produce end-functional glycopolymers with an excellent polydispersity index. The polymers allowed terminal modification with a fluorescent dye by click chemistry. The glycopolymers can dissociate Siglec-7-GD3 interactions at low concentrations.

DOI： 10.1039/c6cc07115e

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Language：English   Publishing type：Part of collection (book)   Publisher：ELSEVIER ACADEMIC PRESS INC  

Neurotrophins are well-characterized neurologically active molecules in the central nervous system. The regulation of these signaling molecules, which are involved in cell growth, differentiation, and survival, is critical for normal brain function. Among the different types of neurotrophins, brain-derived neurotrophic factor (BDNF) is involved in various brain functions, including memory consolidation, synaptic plasticity, and adult neurogenesis, and is therefore a key molecule for understanding comprehensive brain function and neurodevelopmental and psychiatric diseases. The concentration of BDNF in body fluid is highly related to several neurodevelopmental and psychiatric diseases, including Alzheimer's diseases, depression, schizophrenia, and bipolar disorder. In the present review, the mechanisms by which BDNF is released from secretory vesicles are reviewed, with a particular focus on the recently described glycan-mediated release. In addition, the impact of glycan-mediated BDNF release on psychiatric disorders is also discussed.

DOI： 10.1016/bs.vh.2016.11.004

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DOI： doi: 10.1093/glycob/cww128.

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Polysialic acid (polySia) is a linear homopolymer of sialic acid and mainly modifies neural cell adhesion molecule. PolySia plays important roles in synapse formation, learning and memory, social behavior and is associated with several diseases. Gene analyses of one of the biosynthetic enzymes for polySia, ST8SIA2, have revealed that several SNPs and genetic variations in the ST8SIA2 gene are associated with several psychiatric disorders; however, the mechanisms underlying these associations remain unknown. Here, we analyzed the effects of two iSNPs of ST8SIA2, rs2168351 and rs3784730, which are associated with bipolar disorder and autism spectrum disorder, respectively, on the expression of mRNA, ST8SIA2 and its final product, polySia in mouse neuroblastoma and human adenocarcinoma cell lines. We found that both iSNPs affected the expression of pre-mRNA and mRNA of ST8SIA2, and altered the cellular levels of ST8SIA2 and polySia. Taken together, these results indicate that impairment of the regulated expression of ST8SIA2 and the resulting downstream effects on gene products by these two iSNPs contribute to the development of these psychiatric disorders. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2016.08.079

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DOI： doi: 10.1016/j.bbrc.2016.08.079.

Publishing type：Research paper (scientific journal)   Publisher：Biochimica et Biophysica Acta - General Subjects  

DOI： 10.1016/j.bbagen.2016.04.015

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DOI： doi: 10.1016/j.bbagen.2016.04.015.

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DOI： 10.1016/j.bbagen.2016.04.015

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DOI： 10.1016/j.bbagen.2016.04.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Polysialic acid (polySia, PSA) is a unique and functionally important glycan, particularly in vertebrate brains. It is involved in higher brain functions such as learning, memory, and social behaviors. Recently, an association between several genetic variations and single nucleotide polymorphisms (SNPs) of ST8SIA2/STX, one of two polysialyltransferase genes in vertebrates, and psychiatric disorders, such as schizophrenia (SZ), bipolar disorder (BD), and autism spectrum disorder (ASD), was reported based on candidate gene approaches and genome-wide studies among normal and mental disorder patients. It is of critical importance to determine if the reported mutations and SNPs in ST8SIA2 lead to impairments of the structure and function of polySia, which is the final product of ST8SIA2. To date, however, only a few such forward-directed studies have been conducted. In addition, the molecular mechanisms underlying polySia-involved brain functions remain unknown, although polySia was shown to have an anti-adhesive effect. In this report, we review the relationships between psychiatric disorders and polySia and/or ST8SIA2, and describe a new function of polySia as a regulator of neurologically active molecules, such as brain-derived neurotrophic factor (BDNF) and dopamine, which are deeply involved in psychiatric disorders. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2016.04.015

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DOI： 10.1016/j.bbagen.2016.04.015

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Precise directional control of pollen-tube growth by pistil tissue is critical for successful fertilization of flowering plants [1-3]. Ovular attractant peptides, which are secreted from two synergid cells on the side of the egg cell, have been identified [4-6]. Emerging evidence suggests that the ovular directional cue is not sufficient for successful guidance but that competency control by the pistil is critical for the response of pollen tubes to the attraction signal [1, 3, 7]. However, the female molecule for this competency induction has not been reported. Here we report that ovular methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. We developed a method for assaying the response capability of a pollen tube by micromanipulating an ovule. Using this method, we showed that pollen tubes growing through a cut style acquired a response capability in the medium by receiving a sufficient amount of a factor derived from mature ovules of Torenia. This factor, named AMOR, was identified as an arabinogalactan polysaccharide, the terminal 4-O-methyl-glucuronosyl residue of which was necessary for its activity. Moreover, a chemically synthesized disaccharide, the β isomer of methyl-glucuronosyl galactose (4-Me-GlcA-β-(1→6)-Gal), showed AMOR activity. No specific sugar-chain structure of plant extracellular matrix has been identified as a bioactive molecule involved in intercellular communication. We suggest that the AMOR sugar chain in the ovary renders the pollen tube competent to the chemotropic response prior to final guidance by LURE peptides.

DOI： 10.1016/j.cub.2016.02.040

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DOI： 10.1021/acs.orglett.6b00171

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

Very little knowledge has been available on structure and function of heavily glycosylated proteins, whose glycan parts amount to more than 90 % of the whole molecules, mainly because of technical difficulties in detection and determination of structure. In 2004 flagellasialin was discovered as a major cell surface component in the lipid rafts of sea urchin sperm. The extremely high content of glycan chains completely prevented detecting this glycoprotein by conventional protein staining methods, such as Coomassie Brilliant Blue and silver staining. To make this glycoprotein visible, monoclonal antibodies 4F7 and 3G9 were developed as specific probes for detecting glycan chains of flagellasialin. The frequent occurrence of GPI-anchored proteins in lipid rafts is well recognized. Flagellasialin was actually demonstrated to contain a GPI-anchor, based on three lines of experiments. In addition to the computational predictions from the cDNA sequence and the phosphatidylinositolspecific phospholipase C treatment of the sperm membrane fraction, nitrous acid deamination of a flagellasialin-blotted membrane was newly introduced. The immunostaining of flagellasialin on the membrane disappeared after the nitrous acid treatment of the blotted membrane. This method was easy and effective for identifying flagellasialin as a GPI-anchored protein.

DOI： 10.1007/978-4-431-54841-6_167

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Polysialic acid (polySia), particularly α2,8-linked polyNeu5Ac (DP ¼ 8 ~ 400), is a unique polymer of sialic acid (Sia) and modifies neural cell adhesion molecule (NCAM) spatiotemporally in embryonic brains. PolySia is involved in cell migration, neural outgrowth, axonal guidance, synaptic plasticity, and the development of normal neural circuits and neurogenesis due to its anti- adhesive property and spatiotemporal expression. Recent improvements of polySia-detection methods have told us that polySia expression persists in certain areas of the adult brain, such as the olfactory bulb, hippocampus, subventricular zone, thalamus, prefrontal cortex, and amygdala, where neural plasticity, remodeling of neural connections, or neural generation is ongoing. Importantly, several lines of evidence have raised a new concept of polySia function that polySia works as an attractive field that associates with particular ion channels and neurologically active molecules, such as neurotrophic factor (BDNF), growth factor (FGF2), and neurotransmitter (dopamine), to regulate their involved signaling. It has been shown that polySia is involved in learning, memory, circadian rhythm, and social behaviors using polySia-impaired mice. In addition, SNPs and some deletions in genes encoding the polysialyltransferase and NCAM have been reported from patients of schizophrenia, mood disorder and autism. Such impairments of polySia structure derived from polysialyltransferase gene alterations might influence the polySia function not only as the repulsive field but also as the attractive field.

DOI： 10.1007/978-4-431-54841-6_117

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It has been shown that polysialic acid (polySia) is an indispensable component for viability of mouse during development. Especially, it is deeply involved in normal brain function in mouse and human, affecting cell adhesion, cell migration, cell growth, neurogenesis, and synaptogenesis. PolySia is synthesized by two polysialyltransferases, ST8SIA2 and ST8SIA4. Mice deficient in both enzymes show lethality soon after birth. Mice deficient in either ST8SIA2 or ST8SIA4 show significant impairments in learning, memory, circadian rhythm, and social behavior. Interestingly, different phenotypes are often observed between ST8SIA2- and ST8SIA4-deficient mice. Therefore, characterization of these enzymes in vitro and in vivo and precise understanding of the structurefunction relationship of polySia become more and more important. In this chapter, assay procedures for in vitro activity of the polysialyltransferases are focused on to obtain clear results of their in vitro activities that are often said to be very weak compared with other sialyltransferases.

DOI： 10.1007/978-4-431-54841-6_118

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Language：English   Publishing type：Part of collection (book)   Publisher：SPRINGER INT PUBLISHING AG  

Although the structural diversity of sialic acid (Sia) is rapidly expanding, understanding of its biological significance has lagged behind. Advanced technologies to detect and probe diverse structures of Sia are absolutely necessary not only to understand further biological significance but also to pursue medicinal and industrial applications. Here we describe analytical methods for detection of Sia that have recently been developed or improved, with a special focus on 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac), N-glycolylneuraminic acid (Neu5Gc), deaminoneuraminic acid (Kdn), O-sulfated Sia (SiaS), and di-, oligo-, and polysialic acid (diSia/oligoSia/polySia) in glycoproteins and glycolipids. Much more attention has been paid to these Sia and sialoglycoconjugates during the last decade, in terms of regulation of the immune system, neural development and function, tumorigenesis, and aging.

DOI： 10.1007/128_2013_458

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Thyroid-stimulating hormone (TSH; thyrotropin) is a glycoprotein secreted from the pituitary gland. Pars distalis-derived TSH (PD-TSH) stimulates the thyroid gland to produce thyroid hormones (THs), whereas pars tuberalis-derived TSH (PT-TSH) actsonthe hypothalamus to regulate seasonal physiology and behavior. However, it had not been clear how these two TSHs avoid functional crosstalk. Here, we show that this regulation ismediated by tissue-specific glycosylation. Although PT-TSH is released into the circulation, it does not stimulate the thyroid gland. PD-TSH is known to have sulfated biantennary N-glycans, and sulfated TSH is rapidly metabolized in the liver. In contrast, PT-TSH has sialylated multibranched N-glycans; in the circulation, it forms the macro-TSH complex with immunoglobulin or albumin, resulting in the loss of its bioactivity. Glycosylation is fundamental to a wide range of biological processes. This report demonstrates its involvement in preventing functional crosstalk of signaling molecules in the body.

DOI： 10.1016/j.celrep.2014.10.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments. (C) 2014 Elsevier Inc. All rights reserved

DOI： 10.1016/j.bbrc.2014.05.137

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/978-4-431-54240-7_77

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press Inc.  

Frontal affinity chromatography (FAC) is a simple and effective method that is applicable to the analysis of interactions between glycans and glycan-recognition proteins, including lectins, with weak affinity ranging from 10&lt
sup&gt
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to 10&lt
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(M) in terms of dissociation constant (K &lt
inf&gt
d&lt
/inf&gt
). Using conventional instruments, such as a high-performance liquid chromatography (HPLC) system equipped with pump, injector, (fluorescent) detector, and data recorder, the dissociation constants for weak glycan-based interactions can be easily determined with high throughput and accuracy. Notably, if the glycans are labeled with fluorescent dyes, only a small amount of glycans is required for the analysis. Fluorescent labeling of glycans is a common technique, and an increasing number of fluorescent-labeled glycans are commercially available. In this chapter, an advanced FAC method using fluorescent-labeled glycans is described. © 2014 Springer Science+Business Media New York.

DOI： 10.1007/978-1-4939-1292-6_22

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Oligo/polysialic acids consisting of consecutive alpha(2,8)-linkages on gangliosides and glycoproteins play a role in cell adhesion and differentiation events in a manner that is dependent on the degree of polymerization (DP). Anti-oligo/polysialic acid antibodies often have DP-dependent antigenic specificity, and such unique antibodies are often used in biological studies for the detection and differentiation of oligo/polysialic acids. However, molecular mechanisms remain unclear. We here use NMR techniques to analyze the binding epitopes of the anti-oligo/polysialic acid monoclonal antibodies (mAb) A2B5 and 12E3. The mAb A2B5, which has a preference for trisialic acid, recognizes sialic acid residues at the non-reducing terminus and those in nascent units. On the other hand, mAb 12E3, which prefers oligo/polysialic acids of more than six sugar units, recognizes inner sialic acid residues. In both structural complexes, the interresidue transferred NOE correlations are significantly different from those arising from analogs of the free states, indicating that the bound and free sugar conformations are distinct. The ability of the two mAbs to distinguish the chain lengths comes from different binding epitopes and possibly from the conformational differences in the oligo/polysialic acids. Information on the recognition modes is needed for the structural design of immunoreactive antigens for the development of high-affinity anti-polysialic acid antibodies and of related vaccines against pathogenic, polysialic acid-coated bacteria. (c) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2013.07.023

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2174/9781608053865113010005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Sialic acids (Sia) are involved in many biological activities and frequently exist as monosialyl residues at the non-reducing terminal end of glycoconjugates. Occasionally, polymerized structures in the form of disialic acid (diSia), oligosialic acid (oligoSia) and polysialic acid (polySia) are also found in glycoconjugates. In particular, polySia, which is an evolutionarily conserved epitope from sea urchin to humans, is one of the most biologically important glycotopes in vertebrates. The biological functions of polySia, especially on neural cell adhesion molecules, have been well studied and an in-depth body of knowledge concerning polySia has been accumulated. However, considerably less attention has been paid to glycoproteins containing di- and oligoSia groups. However, advances in analytical methods for detecting oligo/polymerized structures have allowed the identification and characterization of an increasing number of glycoproteins containing di/oligo/polySia chains in nature. In addition, sophisticated genetic techniques have also helped to elucidate the underlying mechanisms of polySia-mediated activities. In this review, recent advances in the study of the chemical properties, distribution and functions of di-, oligo- and polySia residues on glycoproteins are described.

DOI： 10.1093/jb/mvt057

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both Nand O-glycans consisting of neutral monosaccharides. Interestingly. WGA-gp may be involved in intracellular Ca2+ regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca2+ oscillation without changing the basal intracellular Ca2+ concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca2+ concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca2+ regulation may be a common feature among animal sperm. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.08.090

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Symposium on the chemistry of natural products  

Gangliosides are a family of glycolipids, which possess sialic acids that play important roles in biological events on cell surfaces. Ganglio-series gangliosides are composed of the tetrasaccharide, (Galβ(1,3)GalNAcβ(1,4)Galβ(1,4)Glc), which bears α(2,8) oligosialic acid units. Glycolipids that vary in number of sialic acids are known as the a-, b-, and c-series of gangliosides. Chemical synthesis of these di-/oligo-sialo-containing glycolipids and their derivatives would facilitate identification of their biological roles. However, the α(2,8) oligosialic acid unit and the compact and rigid 3,4 dibranched galactoside unit are difficult to prepare. Therefore, an effective method for the synthesis of a-, b-, and c-series of gangliosides is still needed. In this report, we describe combinatorial synthesis of ganglio-series ganglioside epitopes varying at the number of the sialic acids. The α(2,3) and α(2,8) sialylations were accomplished by use of 5N,4O-carbonyl and 7,8-O-isopropyliden, and 5N,4O-carbonyl- and 7,8-di-O-chloroacetyl-protected sialyl donors in good yields with excellent a-selectivity, respectively. The two sialyl donors enable synthesis of the di- and tri-sialylgalactosides by simple glycosylation and deprotection. We successfully prepared the ganglioside epitopes via direct construction of a compact, rigid, branched structure. We also reported on a competitive inhibiting activity of the soluble glycolipid library to binding solid-supported GD3 with Siglec-7.

DOI： 10.24496/tennenyuki.54.0_151

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CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of alpha 2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze alpha 2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.

DOI： 10.1074/jbc.M112.365056

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.

DOI： 10.1074/jbc.M111.324186

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Polysialic acid (polySia) is a unique polysaccharide that modifies neural cell adhesion molecule (NCAM) spatiotemporally. Recently, we demonstrated that polySia functions as a reservoir for several neurotrophic factors and neurotransmitters. Here, we showed the direct interaction between polySia and fibroblast growth factor-2 (FGF2) by native-PAGE, gel filtration, and surface plasmon resonance. The minimum chain length of polySia required for the interaction with FGF2 was 17. Compared with heparan sulfate, a well known glycosaminoglycan capable of forming a complex with FGF2, polySia formed a larger complex with distinct properties in facilitating oligomerization of FGF2, as well as in binding to FGF receptors. In polySia-NCAM-expressing NIH-3T3 cells, which were established by transfecting cells with either of the plasmids for the expression of the polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST that can polysialylate NCAM, FGF2-stimulated cell growth, but not cell survival, was inhibited. Taken together, these results suggest that polySia-NCAM might be involved in the regulation of FGF2-FGF receptor signaling through the direct binding of FGF2 in a manner distinct from heparan sulfate.

DOI： 10.1074/jbc.M111.276618

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Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Part of collection (book)   Publisher：John Wiley and Sons  

DOI： 10.1002/9781118017586.ch2

Scopus

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

PubMed

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (international conference proceedings)  

Sialic acids are a family of nine-carbon carboxylated sugars having a nonulosonate skeletal structure (Fig. 35.1). This structure is uniquely different from that of other sugar units of animal glycans. The most popular sialic acid is N-acetylneuraminic acid (Neu5Ac), which is universally found on cell surface glycocalyx and in secreted glycoproteins of vertebrates and some invertebrates. Sialic acids have low acid-base dissociation constants and give a negative charge on the cell surface under a wide range of physiological pH. In nature, more than 50 kinds of sialic acids are known. Nearly all of them are derived from Neu5Ac by a substitution on the hydroxyl groups (e.g., O-acetyl-Neu5Ac) and/or a hydroxylation of the N-acetyl group (e.g., N-glycolylneuraminic acid, Neu5Gc). Each modified sialic acid has properties different from those of Neu5Ac and is believed to contribute to specific physiological functions. In animal cells, sialic acids are most frequently the terminal sugars of cell surface glycolipids and glycoproteins, and any change that occurs on sialic acids can considerably influence the biological properties of a cell. © 2011 Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-4419-7877-6_35

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC CELL BIOLOGY  

Flagellar movement of the sea urchin sperm is regulated by intracellular Ca2+. Flagellasialin, a polysialic acid-containing glycoprotein, as well as other membrane proteins seems responsible for the Ca2+ control. To elucidate the mechanism of Ca2+ dynamics underlying flagellar movement, we analysed the sperm's mechanosensory behavioural responses by using microtechniques. In sea water containing 10 mM Ca2+, the sperm swim in circular paths. When a mechanical stimulus was applied to the sperm head with a glass microstylus, the sperm showed a series of flagellar responses, consisting of a stoppage of beating (quiescence) and a recovery of swimming in a straight path, followed by swimming in a circular path again; as the result the sperm avoided the obstacle. Ca2+-imaging with Fluo-4 showed that the intracellular Ca2+ was high in the quiescence and gradually decreased after that. The effects of blockers and antibodies against candidate components revealed that the Ca2+ influx was induced by Ca2+ channels and the Ca2+ efflux was induced by a flagellasialin-related Ca2+-efflux system, plasma membrane Ca2+-ATPases and the K+-dependent Na+/Ca2+ exchanger. The results show that the Ca2+-dependent mechanosensory behaviour of the sea urchin sperm is regulated by organized functioning of the membrane environment including the plasma membrane proteins and flagellasialin.

DOI： 10.1247/csf.10020

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The monoclonal antibody mAb.A2B5 is a marker for the detection of oligodendrocyte progenitor cells that differentiate into type-2 astrocytes and oligodendrocytes. It is also a useful antibody for separating these cells from other lineage populations. The epitope of this antibody is considered to be the gangliosides GT3 and GQ1c. In this study, we sought to define more precisely the structure of the epitope. Accordingly, we chemically synthesized defined oligosialic acid structures linked to phosphatidylethanolamine and bovine serum albumin and used these to determine the antigenic specificity. mAb.A2B5 recognized the Neu5Ac alpha 2 -&gt; 8Neu5Ac alpha 2 -&gt; 8Neu5Ac alpha -&gt; structure on both glycolipids and glycoproteins. We then examined whether the mAb.A2B5 epitope exists on glycoproteins in developing mouse brains. Western blot analyses revealed the expression of four glycoproteins reactive with the mAb.A2B5, and their expression was dependent on the stage of neural development. All the immunoreactivity in these glycoproteins with mAb.A2B5 disappeared after sialidase treatment and were resistant to chloroform/methanol extraction. These epitopes were also detected in brain homogenates from both GD3 synthetase-null and GD3/GD2 synthetase double null mice. These findings show that the alpha 2,8-trisialic acid (triSia) unit recognized by mAb.A2B5 resides not only on gangliosides but also on glycoproteins in developing mouse brain. We postulate that the triSia structure on glycoproteins may be involved in oligodendrocyte differentiation, similar to the case with the alpha 2,8-triSia structure on gangliosides. Real time polymerase chain reaction analysis of the developmental expression of all known ST8Sia genes, which are responsible for the biosynthesis of alpha 2,8-linked Sia residues, showed that ST8Sia III gene expression correlated with expression of the triSia epitope. We suggest that ST8Sia III is the principal sialyltransferase responsible for synthesis of the alpha 2,8-triSia units on glycoproteins.

DOI： 10.1093/glycob/cwq049

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The beta(1,2)-xylose- and/or alpha(1,3)-fucose-containing cross-reactive carbohydrate determinants (CCDs) are present in various plant and insect N-glycans, and have been attracted as potential antigens in IgE-mediated allergies and immunologically undesired post-translational products on some recombinant therapeutic proteins. By using ELISA and immunoblotting, CCDs-specific IgG and IgE antibodies from some, but not all, of mice and humans were found to fully retain their binding activity after a typical periodate-treatment to CCDs, which did cause the CCDs&apos; antigenic activity to those from the other mice and rabbits to disappear almost completely. Furthermore, the mouse IgE recognizing the periodate-resistant CCDs induced the CCDs/IgE-dependent degranulation of rat basophilic RBL-2H3 cells. These findings indicate that in some cases CCDs include those dependent of the core trisaccharide more strongly than the terminal xylose and fucose, which might have been screened out in the CCDs analyses based on the loss of antibody-binding by the periodate-treatment. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.01.138

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Language：Japanese   Publishing type：Research paper (scientific journal)  

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Acute-phase proteins are an important marker of inflammation and sometimes have a role in the general defense response towards tissue injury. In the present study, we identified a 32-kDa protein that was immunoreactive with monoclonal antibody 2-4B (mAb.2-4B), which is specific to di/oligoNeu5Gc structures, and that behaved as an acute-phase protein following stimulation with either turpentine oil or lipopolysaccharides. The 32-kDa protein was identified as carbonic anhydrase II (CA-II), based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the purified protein. Mouse and human CA-II was immunoreactive and immunoprecipitated with mAb.2-4B, but contained no sialic acid. In addition to mAb.2-4B, the mAb. S2-566 an antibody specific for diNeu5Ac-containing glycans, recognized the CA-II, whereas an anti-oligo/polysialic acid antibody did not. These results indicate that a part of the CA-II protein structure mimics the disialic acid structure recognized by the monoclonal antibodies. This is the first report that CA-II circulates in the serum following inflammation.

DOI： 10.1093/jb/mvm047

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

The egg envelope, referred to as zona pellucida (ZP) in mammalian eggs, is a fibrous and noncollagenous extracellular matrix surrounding vertebrate eggs, and composed of three to four homologous glycoproteins with a common ZP domain. In birds, a liver-derived ZP glycoprotein (ZP1/ZPB1) is transported through the bloodstream to ovarian follicles and joins the egg-envelope matrix construction together with the other ZP glycoproteins, such as ZPC and ZPD/ZPX2, both secreted from follicular granulosa cells. We report here that, through its ZP domain, ZPB1 specifically associates with ZPC, which might lead to the construction of egg-envelope matrix. The ZPB1 in laying hen's serum specifically bound to ZPC, but not to ZPX2, separated by SDS-PAGE and blotted on a membrane. Hemagglutinin (HA)-tagged ZPC expressed in a mammalian cell line (COS-7) cells was processed and secreted as a mature-form into the culture medium. From the culture supernatant of ZPC-expressing transfectants cultured in the presence of ZPB1, both ZPB1 and ZPC were recovered as heterocomplexes by immunoprecipitation using either anti-HA or anti-ZPB1 antibody. Interestingly, a monoclonal antibody, 8E1, which immunoprecipitated free ZPB1, did not immunoprecipitate the ZPB1-ZPC heterocomplexes. An 8E1 epitope was mapped on a C-terminal region of the ZP domain in a ZPB1 molecule by identifying an 8E1-positive peptide using mass spectroscopy. Furthermore, by laser scanning confocal microscopy, ZPB1 and ZPC were observed to colocalize on the surface of ZPC-expressing transfectants cultured in the presence of ZPB1, whereas almost no ZPC was detected on the surface of the transfectants cultured in the absence of ZPB1. Taken together, these results suggest that ZPB1 transported into ovarian follicles encounters and associates with ZPC secreted from granulosa cells, resulting in the formation of heterocomplexes around an oocyte. In addition, it appears that such ZPB1-ZPC complexes accumulated on the oocyte surface act as a scaffold for subsequent matrix construction events including ZPX2 association.

DOI： 10.1095/biolreprod.106.056267

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Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Acute phase proteins are an important marker of inflammation and sometimes have a role in the general defense response towards tissue injury. In the present study, we identified a 32 kDa protein that was immunoreactive with monoclonal antibody 2-4B (mAb.2-4B), which is specific to di/oligoNeu5Gc structures, and that behaved as an acute phase protein following stimulation with either turpentine oil or lipopolysaccharides. The 32 kDa protein was identified as carbonic anhydrase II (CA-II), based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the purified protein. Mouse and human CA-II was immunoreactive and immunoprecipitated with mAb.2-4B, but contained no sialic acid. In addition to mAb.2-4B, the mAb. S2-566 an antibody specific for diNeu5Ac-containing glycans, recognized the CA-II, whereas an anti-oligo/polysialic acid antibody did not. These results indicate that a part of the CA-II protein structure mimics the disialic acid structure recognized by the monoclonal antibodies. This is the first report that CA-II circulates in the serum following inflammation.

Language：Japanese  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

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DOI： 10.1016/j.neuron.2006.10.035

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

A novel alpha 2,9-linked polysialic acid (polySia)-containing glycoprotein of sea urchin sperm flagella was identified and named "flagellasialin." Flagellasialin from Hemicentrotus pulcherrimus shows a diverse relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 40-80 kDa. Flagellasialin is a 96-amino acid, threonine-rich, heavily O-glycosylated (80-90% by weight) glycoprotein with a single transmembrane segment at its C-terminus and no apparent cytosolic domain. Of 12 extracellular Thr residues, eight are O-glycosylated and three are nonglycosylated. Flagellasialin is highly expressed in the testis but cannot be detected in the ovary. The amino acid sequences of flagellasialin from three sea urchin species (H. pulcherrimus, Strongylocentrotus purpuratus, and Strongylocentrotus franciscanus) are identical, but some species differences exist in the three core glycan structures to which the sulfated alpha 2,9-linked polyNeu5Ac chain is linked. Finally, the treatment of sperm with a specific antibody against the alpha 2,9-linked polyNeu5Ac structure results in the elevation of intracellular Ca2+ and inhibition of sperm motility and fertilization, implicating flagellasialin as a regulator of these critical processes.

DOI： 10.1093/glycob/cwl036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Serum glycoproteins are involved in various biologic activities, such as the removal of exogenous antigens, fibrinolysis, and metal transport. Some of them are also useful markers of inflammation and disease. Although the amount of sialic acid increases following inflammation, little attention has been paid to the presence of linkage-specific epitopes in serum, especially the alpha 2,8-linkage. In a previous study, we demonstrated that four components in mouse serum contain alpha 2,8-linked disialic acid (diSia), based on immunoreactivity with monoclonal antibody 2-4B, which is specific to N-glycolyl-neuraminic acid (Neu5Gc)alpha 2 -&gt; (8Neu5Gc alpha 2 -&gt;)(n-1), n &gt;= 2 [Yasukawa et al., (2005) Glycobiology, 15, 827-837]. In this study, we purified three components, 30-, 70-, and 120-kDa gp, and identified them as an immunoglobulin (Ig) light chain, vitronectin, and plasminogen, respectively, using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses. Modifications of these proteins with alpha 2,8-linked diSia were chemically confirmed by fluorometric C-7/C-9 analyses and mild acid hydrolysates-fluorometric anion-exchange chromatography analyses. We also demonstrated that the IgG, IgM, and IgE light chains are commonly modified with alpha 2,8-linked diSia. In addition, both mouse and rat vitronectin contained diSia, and the amount of disialylation in vitronectin dramatically decreased after hepatectomy. These results indicate that a novel diSia modification of serum glycoproteins is biologically important for immunologic events and fibrinolysis.

DOI： 10.1093/glycob/cwj112

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

A novel α2,9-linked polysialic acid-containing glycoprotein of sea urchin sperm flagella was previously identified and named “flagellasialin". Flagellasialin from Hemicentrotus pulcherrimus shows a diverse relative molecular mass on SDS-PAGE of 40-80 kDa. Its glycan structures were determined as HSO3→8Neu5Acα2→9(Neu5Acα2→9)n-2Neu5Acα2→6GalNAcα1→Thr (n, averaging 15) [Miyata et al. (2004) Glycobiology, 14, 827-840]. In this study, we purified flagellasialin, and cloned its cDNA. Flagellasialin is a 96-amino acid, threonine-rich, heavily O-glycosylated (80-90% by weight) glycoprotein with a single transmembrane segment at its C-terminus and no apparent cytosolic domain. Of 12 extracellular Thr residues, eight are O-glycosylated and three are non-glycosylated. Flagellasialin is highly expressed in testis, but cannot be detected in ovary. The amino acid sequences of flagellasialin from three sea urchin species (H. pulcherrimus, Strongylocentrotus purpuratus and S. franciscanus) are identical, but some species differences exist in the three core glycan structures to which the sulfated α2,9-linked polyNeu5Ac chain is linked. Finally, a specific antibody against the α2,9-linked polyNeu5Ac structure inhibits sperm motility and reduces fertilization, implicating flagellasialin as a regulator of these critical processes.

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Serum glycoproteins are involved in various biological activities such as removal of exogenous antigen, fibrinolysis, transport of metal. They are also useful markers of inflammation and diseases. Although it is known that the amount of sialic acid increases after inflammation, little attention has been paid to the presence of linkage specific epitope in serum,especially, the α2,8-linkage. In the previous study, we demonstrated that four components in mouse serum contain the α2,8-linked disialic acid, based on the immunoreactivity with mAb.2-4B that is specific to Neu5Gcα2→(8Neu5Gcα2→)n-1, n≥2 (Yasukawa, Z. et al. (2005) Glycobiology 15, 827-837). In this study, we purified three components, 30kDa-gp, 70kDa-gp, and 120kDa-gp and identified as the light chain of immunoglobulins, vitronectin, and plasminogen, respectively, using MALDI-TOF MS analyses. The modifications of these proteins with the α2,8-linked disialic acid was chemically confirmed by fluorometric C7/C9 analyses and acid hydrolysates-DMB methods. We also showed that the light chains of IgG, IgM and IgE are commonly modified with the α2,8-linked disialic acid. In addition, not only mouse vitronectin but also rat vitronectin was also modified with disialic acid and the amount of disialylation on vitronectin decreased after hepatectomy. These results indicate that disialic acid-modification on serum glycoprotein has some biological meanings in immunological events and fibrinolysis.

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The expression of acute-phase serum proteins increases in response to inflammatory stimuli. Most of these proteins are glycoproteins that often contain sialic acids (Sia). It is unknown, however, how the expression of Sia in these glycoproteins changes during inflammation. This study demonstrates changes in the alpha 2,3-, alpha 2,6-, and alpha 2,8-Sia glycotopes on serum glycoproteins in response to turpentine oil-induced inflammation, based on lectin- and immunoblot analyses by using sialyl linkage-specific lectins, Maackia amurensis for the alpha 2,3-Sia glycotope and Sambucus sieboldiana for the alpha 2,6-Sia glycotopes, and monoclonal antibody 2413 (mAb.2-413) recognizing the di- and oligomers of the alpha 2,8-Neu5Gc residue. There was an increase in a limited number of sialoglycoproteins containing the alpha 2,3-, alpha 2,6-, or alpha 2,8-Sia glycotopes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the expression profiles of mRNAs for the known sialyltransferases in mouse liver during inflammation indicated the up-regulated expression of P-galactoside alpha 2,3-sialyltransferases (ST3Gal I and ST3Gal III) and P-N-acetylgalactosaminide alpha 2,6-sialyltransferase (ST6GalNAc VI) as well as P-galactoside alpha 2,6-sialyltransferase (ST6Gal 1) mRNAs. Notably, ST3Gal I and III and ST6GalNAc VI are involved in the synthesis of the alpha 2,3- and alpha 2,6-Sia glycotopes on O-glycan chains and possibly on gangliosides, whereas ST6Gal I is specific for N-glycan chains. These results provide evidence for the inflammation-induced expression of sialyl glycotopes in serum glycoproteins. We demonstrated that inflammation significantly increased the expression of an unknown 32-kDa glycoprotein containing the alpha 2,8-Sia glycotope. The mechanism for the increase in glycoprotein in inflamed mouse serum remains to be examined, as mRNA expression for all of the alpha 2,8-sialyltransferases (ST8Sia I-VI) was unchanged during inflammation.

DOI： 10.1093/glycob/cwi068

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DOI： 10.1107/S1744309105011577

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Distal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with early adult onset, displays distal dominant muscular involvement and is characterized by the presence of numerous rimmed vacuoles in the affected muscle fibers. The pathophysiology of DMRV has not been clarified yet, although the responsible gene was identified as that encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase involved in the biosynthesis of sialic acids. To identify defective carbohydrate moieties of muscular glycoproteins; from DMRV patients, frozen skeletal muscle sections from seven patients with DMRV, as well as normal and pathological controls, were treated with or without sialidase or N-glycosidase F followed by lectin staining and lectin blotting analysis. The sialic acid contents of the O-glycans in the skeletal muscle specimens from the DMRV patients were also measured. We found that Aracbis hypogaea agglutinin (PNA) lectin reacted strongly with sarcolemmal glycoproteins in the DMRV patients but not with those in control subjects. a-Dystroglycan from the DMRV patients strongly associated with PNA lectin, although that from controls did not. The sialic acid level of the O-glycans in the DMRV muscular glycoproteins with molecular weights of 30 to 200 kd was reduced to 60 to 80% of the control level. The results show that impaired sialyl O-glycan formation in muscular glycoproteins, including a-dystroglycan, occurs in DMRV.

DOI： 10.1016/S0002-9440(10)62332-2

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DOI： 10.1002/jmri.20251

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A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoncuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP + Sia --&gt; CMP-Sia + pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia --&gt; pyruvate + ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate + NADH --&gt; lactate + oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2004.10.033

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A new type of polysialic acid (polySia) structure was demonstrated to occur in a major unknown sialoglycoprotein with a diverse molecular mass of 40-80 kDa in sea urchin sperm. The polySia-containing glycan structure was determined to be HSO3--&gt;8Neu5Acalpha2--&gt;9(Neu5Acalpha2--&gt;9)(n-2) Neu5Acalpha2--&gt;6GalNAcalpha1--&gt;Ser/Thr (n, on average 15), based on carbohydrate analysis of the sialoglycopeptide obtained by an exhaustive protease digestion of whole sperm, fluorometric anion-exchange high-performance liquid chromatography, and methylation analysis. The sulfate group was predominantly localized to the nonreducing terminus of the polySia chain. This is the first example of an alpha2,9-linked polySia structure in animal sperm. The polySia-containing sialoglycoprotein was present in sperm flagellum but not in the head. Furthermore, this sialoglycoprotein localized in the sperm lipid raft, which contains an enriched ganglioside (Neu5Acalpha2--&gt;8Neu5Acalpha2--&gt;6GlcCer), a receptor for sperm-activating peptide (speract), and its associated guanylate cyclase (Ohta et al. [2000] Glycoconj. J., 17, 205-214).

DOI： 10.1093/glycob/cwh100

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A new type of polysialic acid (polySia) structure was demonstrated to occur in a major unknown sialoglycoprotein with a diverse molecular mass of 40-80 kDa in sea urchin sperm. The polySia-containing glycan structure was determined to be HSO3→8Neu5Acα2→9(Neu5Acα2→9)n-2 Neu5Acα2 →6GalNAcα1→Ser/Thr (n, on average 15), based on carbohydrate analysis of the sialoglycopeptide obtained by an exhaustive protease digestion of whole sperm, fluorometric anion-exchange high performance liquid chromatography, and methylation analysis. role in sperm activation and migration in the extracellular coats at fertilization, like polySia on neural cell adhesion molecule acts as a regulator of neural cell functions.

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The 350-kDa sperm-binding protein (SBP), a species-specific sperm-binding protein, is localized in the vitelline layer of sea urchin eggs. In this study, we have shown for the first time that sperm gangliosides are ligands for the intact glycosylated SBP. Using recombinant fragments of the SBP, the N-terminal heat shock protein 110-like domain was shown to be responsible for the binding. The intact SBP could bind various gangliosides, and the binding was sialidase-sensitive and inhibited by sialyllactose, thus indicating that it is the sialic acid-binding protein. Calcium and magnesium ions were not required but they did enhance the binding activity of SBP. The observation that bacterially expressed recombinant SBP and the sialidase-treated intact glycosylated SBP lost divalent cation-dependent enhancement of binding activity suggests that the sialylated carbohydrate moieties of the SBP may be involved in this property. Furthermore, the SBP was shown to bind sperm lipid rafts, in which gangliosides are enriched, and this binding was lost upon sialidase treatment of the lipid rafts. Finally, liposomes containing the ganglioside specifically inhibited fertilization. Taken together, these results allow us to identify SBP as a member of a new class of sialic acid-binding lectin belonging to the Hsp110 family, and indicate that SBP may be involved in interaction of sperm with the vitelline layer of the egg.

DOI： 10.1074/jbc.M307493200

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The neural cell adhesion molecule and the voltage-sensitive sodium channel a-subunit are the only two molecules in mammals known to be modified by alpha-2,8-linked polysialic acid (polySia). We found a new polySia-containing glycoprotein in human milk and identified it as CD36, a member of the B class of the scavenger receptor superfamily. The polySia-containing glycan chain(s) were removed by alkaline treatment but not by peptide: N-glycanase F digestion, indicating that milk CD36 contained polySia on O-linked glycan chain(s). Polysialylation of CD36 occurs not only in human milk but also in mouse milk. However, CD36 in human platelets is not polysialylated. PolySia CD36 is secreted in milk at any lactation stage and reaches peak level at 1 month after parturition. Thus, it is suggested that polySia of milk CD36 is significant for neonatal development in terms of protection and nutrition.

DOI： 10.1074/jbc.M300458200

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